Live-cell Imaging of Lysosomal Membrane Permeabilization During Necroptosis.

Necroptosis, a form of regulated necrosis, culminates in cell membrane rupture. Our lab and others have discovered that lysosomal membrane permeabilization (LMP) is an early and crucial event in this process, preceding membrane rupture. Rapid LMP releases potent lysosomal enzymes, particularly proteases, into the cytosol, actively promoting cell death. Live-cell imaging provides an invaluable tool for detecting LMP during necroptosis in real-time. Several fluorescent dyes are highly effective: (1) pH-sensitive LysoTracker dyes track changes in lysosomal pH. A decrease in fluorescence signal indicates a loss of the lysosomal pH gradient, a primary sign of lysosomal dysfunction, which may be a precursor or direct consequence of LMP. (2) Fluorescein-labeled dextran beads are internalized and accumulate in lysosomes. Their release into the cytosol signals complete LMP and cargo leakage. Here, we observed a progressive loss of Lysotracker fluorescence, with diffusing Dextran fluorescence into the cytosol after necroptosis induction. Thus, the live-cell imaging methodology enables researchers to precisely track the timing and extent of lysosomal dysfunction, contributing to a more comprehensive understanding of necroptosis mechanisms and illuminating potential therapeutic interventions.